The establishment of left-right asymmetry is a fundamental process in animal development. Interference with this process leads to a range of disorders collectively known as laterality defects, which manifest as abnormal arrangements of visceral organs. Among patients with laterality defects, congenital heart diseases (CHD) are prevalent. Through multiple model organisms, extant research has established that myosin-Id (MYO1D) deficiency causes laterality defects. This study investigated over a hundred cases and identified a novel biallelic variant of MYO1D (NM_015194: c.1531G>A; p.D511N) in a consanguineous family with complex CHD and laterality defects. Further examination of the proband revealed asthenoteratozoospermia and shortened sperm. Afterward, the effects of the D511N variant and another known MYO1D variant (NM_015194: c.2293C>T; p.P765S) were assessed. The assessment showed that both enhance the interaction with β-actin and SPAG6. Overall, this study revealed the genetic heterogeneity of this rare disease and found that MYO1D variants are correlated with laterality defects and CHD in humans. Furthermore, this research established a connection between sperm defects and MYO1D variants. It offers guidance for exploring infertility and reproductive health concerns. The findings provide a critical basis for advancing personalized medicine and genetic counseling.

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function.

Adult B-cell acute lymphoblastic leukemia (B-ALL) prognosis remains unsatisfactory, and searching for new therapeutic targets is crucial for improving patient prognosis. Sperm-associated antigen 6 (SPAG6), a member of the cancer-testis antigen family, plays an important role in tumors, especially hematologic tumors; however, it is unknown whether SPAG6 plays a role in adult B-ALL. In this study, we demonstrated for the first time that SPAG6 expression was up-regulated in the bone marrow of adult B-ALL patients compared to healthy donors, and expression was significantly reduced in patients who achieved complete remission (CR) after treatment. In addition, patients with high SPAG6 expression were older (≥ 35 years; P = 0.015), had elevated white blood cell counts (WBC > 30 × 109/L; P = 0.021), and a low rate of CR (P = 0.036). We explored the SPAG6 effect on cell function by lentiviral transfection of adult B-ALL cell lines BALL-1 and NALM-6, and discovered that knocking down SPAG6 significantly inhibited cell proliferation and promoted apoptosis. We identified that SPAG6 knockdown might regulate cell proliferation and apoptosis via the transforming growth factor-β (TGF-β)/Smad signaling pathway.

Sperm associated antigen 6 (Spag6) is the PF16 homolog of Chlamydomonas and participates in the regulation of cilia movement. Studies have shown that Spag6 is expressed in the brain, and its loss will lead to cerebral edema caused by a defect in motor cilium function in ependymal cells. However, it has not been reported whether the limited or extensive cerebral edema resulting from ischemic strokes is related to the expression regulation of Spag6. Therefore, this study aimed to investigate the effect and related mechanism of Spag6 in alleviating Cerebral Ischemic stroke-reperfusion (CIS/R) damage. Our experimental results showed that Spag6 overexpression alleviated CIS/R-mediated motor cilia structural disorder, improved cerebral edema, inhibited nerve injuries in rats with cerebral ischemia, and alleviated synaptic and dendritic spinal injuries by regulating the expressions of synaptic-related proteins such as CaMKII, PSD95, and CREB. Based on significant changes in PI3K/AKT-mTOR signaling pathway activity after CIS/R determination, we determined that Spag6 regulates the abnormal expression of CIS/R-induced inflammatory factors NF-κB, NLRP3, IL-10, and the autophagy-related proteins Beclin-1, LC3, and P62 by activating the PI3K/AKT-mTOR signaling pathway. This inhibits inflammation and autophagy in the brain tissue. In summary, this study revealed that Spag6 alleviates brain edema damage after CIS/R by maintaining the structural function of the motor cilium, regulating the PI3K/AKT-mTOR signaling pathway, and inhibiting inflammation and autophagy reaction.

Mouse sperm-associated antigen 6 like (SPAG6L) is an axoneme central apparatus protein, essential for the normal function of the ependymal cell and lung cilia, and sperm flagella. Accumulated evidence has disclosed multiple biological functions of SPAG6L, including ciliary/flagellar biogenesis and polarization, neurogenesis, and neuronal migration. Conventional Spag6l knockout mice died of hydrocephalus, which impedes further investigation of the function of the gene in vivo. To overcome the limitation of the short lifespan of conventional knockout mice, we developed a conditional allele by inserting two loxP sites in the genome flanking exon 3 of the Spag6l gene. By crossing the floxed Spag6l mice to a Hrpt-Cre line which expresses Cre recombinase ubiquitously in vivo, mutant mice that are missing SPAG6L globally were obtained. Homozygous mutant Spag6l mice showed normal appearance within the first week after birth, but reduced body size was observed after 1 week, and all developed hydrocephalus and died within 4 weeks of age. The phenotype mirrored that of the conventional Spag6l knockout mice. The newly established floxed Spag6l model provides a powerful tool to further investigate the role of the Spag6l gene in individual cell types and tissues.

BACKGROUND: DNA methylation controls the transcription of genes and is involved in the development of lung cancer. Our preliminary bioinformatics prediction revealed that sperm associated antigen 6 (SPAG6) was considerably hypermethylated in lung squamous cell carcinoma (LUSC). Thus, this study aimed to probe the mechanism underlying its hypermethylation.
METHODS: The effect of DNA methylation of SPAG6 on its expression in LUSC was analyzed. The contributors to SPAG6 DNA hypermethylation were sought. CCK-8, EdU, and Transwell assays were carried out to assess the malignant phenotype of LUSC cells. KEGG pathway enrichment analysis was used to screen for pathways affected by SPAG6, which were confirmed by dual-luciferase assays. Bioinformatics analysis was conducted to dissect the impact of SPAG6 on the immune response and cancer cell stemness in LUSC.
RESULTS: DNA methyltransferase 3b (DNMT3b)-mediated hypermethylation of the SPAG6 promoter in LUSC led to SPAG6 downregulation. SPAG6 reverted the malignant phenotype of LUSC cells. SPAG6 regulated the JAK/STAT pathway by inhibiting the transcription of STAT1 and STAT3. The expression of SPAG6 was positively related to immune infiltration in LUSC and inversely related to the expressions of the immunosuppressive genes CTLA4 and PDCD1. SPAG6 expression was negatively correlated with cancer cell stemness in LUSC, and its expression inhibited the expressions of Nanog, ALDH1, and Sox2, markers of cancer cell stemness.
CONCLUSIONS: DNMT3b-mediated SPAG6 promoter hypermethylation activates the JAK/STAT pathway to promote LUSC progression.

Objective: To investigate the expression and clinical significance of sperm-associated antigen 6 and NM23 proteins in human osteosarcoma. Methods: The specimens of conventional osteosarcoma with follow-up from 42 Chinese patients were analyzed in this study, and 12 cases of osteochondroma were considered controls. The expression of SPAG6 and NM23 was inspected using immunohistochemical staining, qRT-PCR, and Western blotting methods. Results: The positive expression rate of SPAG6 protein (71.43%) in 42 cases of osteosarcoma tissue was significantly higher than that (33.33%) in 12 cases of osteochondroma tissues (p < 0.05), while the positive rate of NM23 protein (35.71%) in osteosarcoma tissue was lower than that (58.33%) in osteochondroma tissue (p < 0.05). The mRNA and protein levels of SPAG6 were significantly higher than those of the adjacent normal tissues, while the expression of NM23 was lower in osteosarcoma tissues than that in the controls (p < 0.05 for all). There was a positive relationship between the expression of SPAG6 and pathological grade, metastasis, and Enneking stage (p < 0.05 for all). The overall survival rate of osteosarcoma patients with SPAG6 positive expression was significantly lower than that with SPAG6 negative expression. The relationship between the expression of NM23 and pathological grade, metastasis, and Enneking stage was negative (p < 0.05 for all). The overall survival rate of the osteosarcoma patients with NM23 positive expression was higher than that of the patients with NM23 negative expression (p < 0.05). Conclusion: Overexpression of SPAG6 and low expression of NM23 are negatively related to pathological grade, metastasis, and Enneking stage and prognosis of osteosarcoma patients. This suggested that SPAG6 and NM23 should be considered candidate prognostic biomarkers for patients with osteosarcoma.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a subtype of severe asthenoteratozoospermia with poorly understood genetic etiology. SPAG6 is a core axonemal component that plays a critical role in the formation of cilia and sperm flagella. Previous studies have reported that mutations in SPAG6 cause primary ciliary dyskinesia (PCD), but the association between SPAG6 gene variants and the MMAF phenotype has not yet been described.
METHODS: We performed whole-exome sequencing (WES) in two unrelated Han Chinese men with MMAF. Sanger sequencing was used to validate the candidate variants. Routine semen analysis was carried out according to the WHO guidelines (5th Edition). Sperm morphology was assessed using modified Papanicolaou staining. Scanning and transmission electron microscopy (S/TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to examine the expression of SPAG6 protein. Assisted fertilization with intracytoplasmic sperm injection (ICSI) was applied.
RESULTS: Two homozygous SPAG6 variants were identified by WES and Sanger validation in two patients with MMAF phenotype (F1 II-1: c.308C > A, p. A103D; F2 II-1: c. 585delA, p. K196Sfs*6). Semen analysis showed progressive rates of less than 1%, and most of the spermatozoa presented MMAF by Papanicolaou staining. TEM revealed that the overall axonemal ultrastructure was disrupted and primarily presented an abnormal \"9 + 0\" configuration. No other PCD-related symptoms were found on physical examination and medical consultations, as well as lung CT screening. The level of SPAG6 protein was significantly decreased in the spermatozoa, and IF analysis revealed that SPAG6 staining was extremely weak and discontinuous in the sperm flagella of the two patients. Notably, F1 II-1 and his wife conceived successfully after undergoing ICSI.
CONCLUSIONS: Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of Chlamydomonas PF16, an axonemal central pair protein involved in flagellar motility. In mice, two Spag6 genes have been identified. The ancestral gene, on mouse chromosome 2, is named Spag6. A related gene originally called Spag6, localized on mouse chromosome 16, evolved from the ancient Spag6 gene. It has been renamed Spag6-like (Spag6l). Spag6 encodes a 1.6 kb transcript consisting of 11 exons, while Spag6l encodes a 2.4 kb transcript which contains an additional non-coding exon in the 3\'-end as well as the 11 exons found in Spag6. The two Spag6 genes share high similarities in their nucleotide and amino acid sequences. Unlike Spag6l mRNA, which is widely expressed, Spag6 mRNA expression is limited to a smaller number of tissues, including the testis and brain. In transfected mammalian cells, SPAG6/GFP is localized on microtubules, a similar localization as SPAG6L. A global Spag6l knockout mouse model was generated previously. In addition to a role in modulating the ciliary beat, SPAG6L has many unexpected functions, including roles in the regulation of ciliogenesis/spermatogenesis, hearing, and the immunological synapse, among others. To investigate the role of the ancient Spag6 gene, we phenotyped global Spag6 knockout mice. All homozygous mutant mice were grossly normal, and fertility was not affected in both males and females. The homozygous males had normal sperm parameters, including sperm number, motility, and morphology. Examination of testis histology revealed normal spermatogenesis. Testicular protein expression levels of selected SPAG6L binding partners, including SPAG16L, were not changed in the Spag6 knockout mice, even though the SPAG16L level was significantly reduced in the Spag6l knockout mice. Structural analysis of the two SPAG6 proteins shows that both adopt very similar folds, with differences in a few amino acids, many of which are solvent-exposed. These differences endow the two proteins with different functional characteristics, even though both have eight armadillo repeats that mediate protein-protein interaction. Our studies suggest that SPAG6 and SPAG6L have different functions in vivo, with the evolved SPAG6L protein being more important. Since the two proteins have some overlapping binding partners, SPAG6 could have functions that are yet to be identified.

The main purpose of the present study was to elucidate the role of sperm‑associated antigen 6 (SPAG6) in the occurrence and development of Burkitt lymphoma (BL) and explore the underlying molecular mechanisms. A correlation was observed between the expression of SPAG6 and the prognosis of patients with lymphoma using The Cancer Genome Atlas (TCGA) database analysis. It was demonstrated that the levels of SPAG6 in BL cells were higher compared with that in IM‑9 cells by reverse transcription‑PCR and western blot assays. Moreover, silencing of SPAG6 significantly decreased proliferation and increased apoptosis of Daudi and Raji cells, whereas SPAG6 overexpression exerted the opposite effects on CA46 and NAMALWA cells. When investigating the possible mechanism, it was first observed that the level of phosphatase and tensin homolog (PTEN) protein was significantly increased, while that of phosphorylated (p‑)AKT protein was markedly reduced in the SPAG6‑knockdown group compared with the blank control group in Daudi and Raji cells by western blot analysis. It was further ascertained whether the phosphoinositide 3‑kinase (PI3K)/PTEN/protein kinase B (AKT) pathway mediates the effects of SPAG6 on cell proliferation and apoptosis, and the results demonstrated that silencing of SPAG6 suppressed the viability of Daudi and Raji cells, whereas PTEN knockdown using siRNA or SF1670 (a specific PTEN inhibitor) reversed the inhibitory effect on cell proliferation and the promoting effect on cell apoptosis induced by SPAG6 depletion in vitro as well as in vivo. These data revealed that SPAG6 may promote the proliferation and inhibit the apoptosis of BL cells via the PTEN/PI3K/AKT pathway. The results of the present study suggest that SPAG6 may play a key role in the progression of BL and may be of value as a predictive prognostic biomarker in patients with BL.